The function of selenoprotein W is unknown but is a selenocysteine containing protein, with highest concentrations in muscle, testes and brain. Selenium deficiency is associated with a failure to incorporate selenium into this protein, suggesting a critical metabolic role for it. Information on the function of selenoprotein W will be sought with knockout mice. Based on the genomic sequence, a knockout vector will be designed for injection into embryonic stem cells. After established procedures have been used to produce chimeric mice, they will be bred to normal mice to obtain heterozygotes. These will be used in selected breeding to obtain homozygotes which should lack the gene for selenoprotein W. Southern blots will be used to determine if the desired animals have been obtained. Experiments are planned to determine if the knockout mice are more subject to peroxidation, using three different compounds to promote this disorder. The effects of selenoprotein W deletion on the expression of other selenoproteins such as the cellular glutathione peroxidase (GPX1) and the phospholipid hydroperoxidase (GPX4) will be examined in these knockout mice. The knockout mice will be given selenium to show that this element will increase the activities of GPX1 and GPX4 but not selenoprotein W. This will be followed by oxidative stress studies with tissue cultures of muscle cells to confirm the results in the whole animal. Glutathione and a smaller adduct are stoichiometrically bound to selenoprotein W from muscle, and the identification and binding of this small adduct to selenoprotein W will be explored as a means of further investigation into its metabolic function. We have access to knockout mice deficient in gamma-glutamyl transpeptidase and these will be used to determine the effects of altered glutathione and cystine levels on selenoprotein W content. These animals will also be supplemented with selenium to determine the effects upon selenoprotein W and GPX1 and GPX4 induction.